Project Summary Cell polarization is a fundamental feature of many cell types that display specialized morphologies to perform distinct functions. Odontoblasts are a type of highly polarized dental cells with a high secretory function to form dentin that is a major component of a tooth. The polarization of odontoblasts is a prerequisite for the formation of dentin with tubular structure that is crucial for maintaining the normal biological functions of a tooth. Many studies have shown that dental pulp stem cells (DPSCs) can be polarized and differentiated into odontoblast-like cells. However, the factors that control DPSC polarization and the underlying mechanism remain unknown. Because of that, most of tissue engineering approaches for regenerative endodontics only regenerated non-tubular bone-like mineralized tissues. The main obstacle to explore DPSC polarization is the lack of a bio- inspired three-dimensional (3D) ?clean? platform that is capable of deciphering the biophysical and biochemical signals that initiate and regulate DPSC polarization. Recently, we developed a bio-inspired tubular 3D matrix and successfully regenerated highly organized tubular dentin. Furthermore, we identified that the tubular architecture of the synthetic matrix is a crucial biophysical factor to initiate DPSC polarization and form tubular dentin. In our pilot study, we have further developed a unique micropatterning and laser ablation technology to create a bio-inspired 3D ?clean? platform that can precisely manipulate one single cell (or multiple cells) in a microisland of the 3D platform, therefore, is capable of deciphering the signals that initiate/regulate DPSC polarization. The proposed project, therefore, is to use the unique ?clean? 3D platform to identify and analyze the biophysical and biochemical factors that control DPSC polarization. We hypothesize that DPSC polarization is initiated by a set of biophysical and biochemical factors that work synergistically to regulate and stabilize the polarized DPSCs. To accomplish the overall objective for this project, the following two aims are proposed: Aim 1 is to identify biophysical factors that initiate and modulate DPSC polarization; and Aim 2 is to identify biochemical factors that regulate DPSC polarization. Successfully completing this work will fundamentally advance the understanding of DPSC polarization and greatly promote the ability to develop new bio-inspired matrices for regenerative endodontics.